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Objective To construct and evaluate a novel PAMAM dendrimer vector-DNA vaccine for schistosomiasis japonica.Methods Lysine was used to modify 4.0G PAMAM.and the modified product PAMAM-Lys was synthesized.Agarose gel electrophoresis was used to confirm the composite ratio of plasmid DNA and dendrimer.Micrestructure of the compound was observed by using transmission electronic microscopy,and the stability was analyzed by using electrophoresis.The viability of the cells transfected with dendrimers was evaluated by using a MTT technique in vitro.Fiftyty mice were immunized with purified plasmid pJW4303,pJW4303-Sj23 dendrimer PAMAM-Lys and compound PAMAM-Lys/pJW4303-Sj23,respectively.The specific antibodies of the mice in each group were detected to access the immunoreactivity.Results The agarese gel electrophoresis showed that when the charge ratio of the dendrimer vector and DNA was between 2 and 4,the positive and negative charges could be counteraeted completely,and the compound was blocked completely by DNA electrophoresis.The obscrvation results with transmission electronic microscopy showed that the composition of dendrimer vector and DNA caused shrink of DNA structure.Dendrimer-DNA compound had a good stability.MTT showed the modified dendrimer vector and DNA compound system produced a lower cell toxicity on 293T cell than the unmodified Ones.Thk levels of specific antibodies of the mice immunized with PAMAM-Lys/pJW4303Sj23 were significantly higher than those of the mice immunized with naked DNA vaccine(P<0.05).Conclusions The lysinemodified PAMAM-lys is an excellent vector,and has an appropriate biocompatibility.Lysine-modification can reduce the cell toxicity of PAMAM dendrimer significantly.PAMAM-Lys can enhance the immunoreactivity of DNA vacmine which merits further application in schistosomiasis DNA vaccine.
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<p><b>OBJECTIVE</b>To identify the egg antigens related to the formation of hepatic granulomas and fibrosis of Schistosomiasis japonica.</p><p><b>METHODS</b>The egg cDNA library of Schistosoma japonicum (S. japonicum) was constructed and screened by immunological methods with the pooled sera of advanced schistosomiasis patients. The inserted foreign DNA fragments of positive clones were sequenced. The sequence data were analyzed using Wdnasis 2.5 and compared with Genebank data using blast software.</p><p><b>RESULTS</b>Eighty-one clones containing recombinant DNA fragments were obtained from the egg cDNA library of S. japonicum by immunological screening. The DNA sequences of all clones belonged to the miracidial antigen family. The longest cDNA fragment was 1604 bp, which contained an open reading frame of 351 bp, which encoded a protein of 1 2913.35 daltons.</p><p><b>CONCLUSION</b>The cDNA sequence of the miracidial antigen of S. japonicum (Chinese strain) was obtained for the first time.</p>
Assuntos
Animais , Antígenos de Helmintos , Genética , Sequência de Bases , China , DNA Complementar , Óvulo , Schistosoma japonicum , Genética , Alergia e ImunologiaRESUMO
Objective To develop a novel synergism compound suspension concentrate of niclosamide and chlorphoxim (Co-SCN) and sdudy its characteristics. Methods Niclosamide and chlorphoxim were milled by a ball mill and mixed with different amounts of wetting agent. disper-sant agent, thickener, and water etc. , to develop Co-SCN, and the pH value, thickener, grain size were evaluated. The ultraviolet absorption spectrum of niclosamide and chlorphoxim were measured. The content of niclosamide and chlorphoxim in the solution were assayed by HPLC. Results Co-SCN was a gray thickener fluidity liquid. It was very easy to disperse and could be mixed with water in any proportion. Its pH was 8. 65 and thickener was 137 mpa? s. The grain sizes (diameter) were from 0. 138-19. 953 ?m. Of them more than 95. 6% was smaller than 10 ?m and more than 82. 24% was smaller than 5 ?m. There were three peaks of ultraviolet absorption spectrum for niclosamide: 210, 234 nm and 334 nm respectively. One peak of chlorphoxim was at 269 nm. The novel formulation contained 20.64% niclosamide and 5.26% chlorphoxim. The suspension stability of Co-SCN was 100% for 2 hours and 89. 14% for 4 hours, and otherwise WPN in water was speedy sediment. Conclusion The novel synergism compound suspension concentrate of niclosamide and chlorphoxim is a stable quality and standard formulation.
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Objective To evaluate the molluscicidal effect of colistin E on Oncomelania hupensis. Methods The molluscicidal effect of colistin E on O. hupensis was checked by using the immersion method and spraying method. The toxicity of colistin E on fish was observed by using the toxic test. Results The snail mortality of each group was 100% when the snails were immersed in the colistin E solutions at a concentration of 5. 0, 1. 0 g/L for 24, 48 h and 72 h separately. When the snails were immersed in the colistin E solution at a concentration of 0. 5 g/L for 24, 48 h and 72 h, the death rates were 95% , 100% and 100% respectively; when the snails were immersed in the colistin E solution at a concentration of 0. 1 g/L for 24, 48 h and 72 h, the mortality was 90%, 95% and 100% respectively; when the snails were immersed in the colistin E solution at a concentration of 0. 01 g/L for 24, 48 h and 72 h, the mortality was 70%, 86% and 100% respectively. The snail mortality by the spraying method in a dose of 35, 70, 140 mg/m2 of colistin E was 60%, 100% and 100%. The result of toxic test showed that the toxicity of colistin E on fish was low. Conclusion Colistin E is an effective molluscicide, and is worthy of investigation further.
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Objective To evaluate the toxicity of suspension concentrate of niclosamide(SCN)for molluscicide in the field.Methods According to the state standard of the People's Republic of China "The methods of toxicity test for agriculture register",GB15670-1995,the experiments of acute toxicity on rats and fish were carried out.Results LD50(s)of SCN via mouth and skin with rats were more than 5 000 mg/kg respectively,and LC50(s)of SCN via inbreathe with rats were more than 5 000 mg/m3.Based on the classification of appraising criterion on acute toxicity test,it belonged to a feebleness toxicity degree.The eye and the skin stimulating tests with rabbits showed that it did not irritate the eyes and the skin.For fish,its acute toxicity was slightly lower than that of pure niclosamide,and markedly lower than that of pure niclosamide ethanolamine salt and WPN.Conclusions SCN belongs to a feebleness toxicity degree and has a lower toxicity to fish.It should be a useful molluscicide in endemic areas of schistosomiasis.
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Objective To identify the trend of endemic situation among surveillance sites in Jiagsu Province from 2000 to 2002. Methods Twelve schistosomiasis surveillance sites were es-tablished ,and the longitudinal, surveillance was carried out. Results The related index of snail increased in most of surveillance sites, the rates of positive snails rose rapidly in marshlands. The infection rates of Schitosoma janponicum of cattle decreased and infection rates of human were relatively steady. However, there was still the danger of heavy endemic. Conclusion Current control strategies can not effectively adapt to the endemic situation of schistosomiasis, although which have some effects on control of morbidity. We need to study the new characteristics and rule of the endemic of schistosomiasis, and make out more effective control strategies which can suit with the current society, economies and nature environment.
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This study reported the primary application of FA-ELISA from 107-121kDa fration antibody of Schistosoma Japonicum for detection of the short-term antibody in patients with schistosomiasis. The result showed that this method provided high sensitivity (with positive rate of 93. 33% with 30 cases of schistosomiasis) and good specificity (no false postive in 60 health objects). When one group of schistosome patients were examined with FA -ELISA and with the routine SEA-ELISA before chemotherapy and at 8 months,1 year and 1. 5 year after treatment,it showed good property for evaluation of chemotherapy efficacy with FA -ELISA,which was much better than the routine method.
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Objective To screen the bacteria and its components which are toxic to Oncomelania hupensis. Methods The samples of Oncomelania hupensis on the point of death and the soil around the snails were collected. The bacteria existing in the snails and soil were isolated and identified by using regular methods. After being fermented, the toxicity of the bacterium components including the ferment supernatant, split products and bacteria to the snail were tested by the toxicity test. Results Totally, 104 strains of bacteria were isolated from the snails and soil samples, which included Gram positive cocci or bacilli, Gram negative cocci or bacilli. The fermenting supernatant, splitting products and 10 strains of bacteria showed different level of toxicity against the snail respectively. All the fermenting supernatant of bacterium PY1-1, PY7-2, PY3-5, PY19-3, PY2-2-2, PY8-2-2, PY16-1 could kill more than 50 percent of snails. Conclusion The bacteria which are poisonous to snails have been isolated that make a good basis for identifying single toxic component against snails.
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Objective To construct, express and purify human scFv antibody (S1) against the recombinant SAG1 of Toxoplasma fused to magainin, and observe its protective effect against Toxoplasma in infected mice. Methods The S1 scFv antibody gene amplified from phagmid S1/pIT-2 fused to magainin was cloned into procaryotic expression vector pET-32c. The recombinant plasmid S1M/pET-32c proved by DNA sequencing was transformed into E.coli BL21, and induced for fusion expression of S1M with IPTG. The expressed S1M was purified with Ni 2+ chelating HiTrap HP column and detected with SDS-PAGE. The effect of reduction of infection of Toxoplasma was observed through in vivo and in vitro experiments in mice. Results The fused gene of S1 and magainin was successfully cloned into procaryotic expression vector pET-32c proved by DNA sequencing. The recombinant S1M protein about 43 kDa was expressed in E.coli as inclusion body, and prepared with Ni 2+ column purification. Tachyzoite of Toxoplasma preincubated with S1M showed decreased infectivity in mice, the result of in vivo experiments showed that mice treated with S1M hadlonger survival time than the mice untreated. Conclusion The purified targeting antimicrobial peptide S1M could reduce the infectivity of tachyzoites of Toxoplasma in a certain extent and has a potential value for biological therapy of toxoplasmosis; otherwise, the constructed targeting antimic robial peptide S1M also provides a new model for biological therapy of toxoplasmosis.
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Objective To design the Internet geographic information system (GIS) on schistosomiasis of Jiangsu Province using the technology of WebGIS. Methods Based on the GIS database of schistosomiasis, the active model of WebGIS on schistosomiasis was developed with the software of ArcIMS. Results The WebGIS of schistosomiasis has been developed and it has been running on the intranet of Jiangsu Institute of Parasitic Diseases. The system would manage the geographic database and attribute material database, and it would provide the function of query, display, analysis, statistics, export, etc. Conclusion It is feasible to design the WebGIS of schistosomiasis, which provides the basics of developing the Management Decision Systems of Schistosomiasis of Jiangsu Province.
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Objective To understand whether the breeding time of snails influences the evaluation on molluscicidal efficacy against Oncomelania snails so as to make a standardization of methods for screening molluscicides in laboratory. Methods Snails collected from the endemic areas in Nanjing and Tongling cities bred for 1,6,11,16,21.31,61 and 91 days respectively, and then were immersed in 1.000 0,0.500 0,0.250 0,0.125 0,0.062 5 and 0.031 3 mg/L solution of niclosamide for 24 and 48 hours at 25℃ in laboratory. Results One hundred percent killing rate was achieved in the groups of the snails bred for 1 - 11 days and immersed in 1.0 mg/L niclosamide for 24 hours. The LC_(50) concentrations of snails collected from Nanjing, Jiangsu Province and immersed for 24 hours varied from 0.094 7 to 0.133 9 mg/L, and for 48 hours from 0.071 8 to 0.092 6 mg/L.Those of snails collected from Tongling, Anhui Province for 24 hours varied from 0.082 5 to 0.103 9 mg/L, and for 48 hours from 0. 071 8 to 0.082 5 mg/L. The molluscicidal effect was unstable in the groups of snails bred for more than 16 days. There was a significant difference (X_(Nanjing24 h)~2 = 22.26,P
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Objective To analyze the full length cDNA sequence of Schistosoma japonicum egg miracidia genes harboring signal sequence.Methods The gene specific primers were designed and synthesized according to S.japonicum egg miracidia cDNA fragment containing signal sequence identified by signal sequence trapping method previously. The 5′and 3′ end cDNA fragments of each egg miracidia cDNA fragment harboring signal sequence were amplified by nest PCR using the first strand cDNA of S.japonicum as the template. The specific PCR fragments were cloned by TA clone method and sequenced. The full length cDNA sequence of each gene with signal sequence was constructed by comparing the cDNA sequence identified with signal sequence trapping method and the 5′ end sequence, the 3′ end sequence and deleting the overlapping fragments. The splicing model between mRNA of signal sequence and one of mature portions of S.japonicum egg miracidia gene was checked by analyzing the genomic DNA sequence structure of some genes with signal sequence. Results The 5′ and 3′ end cDNA fragments of sixteen among thirty cDNA fragment with signal sequence were amplified successfully, and their DNA sequences were determined. The full length cDNA sequences of sixteen egg miracidia genes were obtained by sequence matching and splicing. The results of deduced amino acid analysis found that the signal peptide of gene SjP4001 was the same to the one of SjP1531 and the signal peptide of gene SjP1183 was similar to the one of gene SjP3742. It confirmed that different genes could share the same or similar signal peptide. The data of S.japonicum genomic DNA sequence analysis showed that the S.japonicum could obtain its signal sequence by alternative splicing model or trans-splicing model. Conclusions The full length cDNA sequences of sixteen S.japonicum egg miracidia genes with signal sequence have been defined, it indicated that the S.japonicum egg miracidia genes could get its signal sequence by alternative splicing model or trans-splicing model was found in this study.
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Objective To predict B cell epitopes in Sj22, Sj23, Sj14-3-3, Sj26 of Schistosoma japonicum with bioinformatics, and evaluate the antigenicity of these epitope proteins. Methods The complete DNA sequences of S.japonicum were predicted by BioSun system, the target B cell epitope genes were selected, cloned and expressed. The expressed fusion proteins were detected with the sera of schistosomiasis patients and health people for evaluation of their antigenicity. Results Eight B cell epitopes from four molecules of S.japonicum were predicted. The B cell epitopes of Sj22 probably located in 56-62 and 127-133 amino acids. The B cell epitopes of Sj23 probably located in 149-156 and 160-167 amino acids. The B cell epitopes of S14-3-3 probably located in 118-125 and 130-137 amino acids. The B cell epitopes of Sj26 probably located in 143-149 and 191-197 amino acids. The predicted epitope genes were cloned into pET-32c plasmid and expressed. Three of eight expressed fusion proteins of epitopes were reacted with the sera of schistosomiasis patients but not with health people. Conclusion Three epitope antigens with potential diagnosis value are determined.
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Objective To evaluate the stability of dipstick dye immunoassay (DDIA) kit forschisitosomiasis diagnosis. Methods By means of detection of the sera from infected people withSchistosoma japonicum and healthy people, the stability of the DDIA kit, which stored at 37℃,room temperature or 4 ℃ respectively, was evaluated depending on the detective results ofsensitivity, specificity, detectable minimum and coefficient variation ( CV). Results Thesensitivity, specificity, detectable minimum and coefficient variation of the DDIA kit were invariableafter the kits stored at 37 ℃ for 180 days, and at room temperature or 4 ℃ for 360 days.Conclusion The DDIA kit is stable while it stores at 37℃ for 180 days, and at room temperatureor 4℃ for 360 days at least.
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Objective To investigate the value of recombinant fusion protein (GST-HD)of the large hydrophilic domain (HD) of 23 kDa membrane protein of Schistosoma japonicum with the Glu-tathione-S-transferase (GST) of S. japonicum for early diagnosis of schistosorniasis. Methods The rabbits were infected with cercariae of S. japonicum Chinese mainland strain (300 per one). The rabbits' sera before infection and after being infected at different time were collected. The antibodies IgG(s) against recombinant GST-HD and SEA were measured respectively by ELISA to observe the rabbits' immune reaction status to GST-HD and SEA at different time after being infected with the cercariae. At the same time, 90 serum samples of patients with acute schistosorniasis and 30 samples of healthy persons were checked with GST-HD to evaluate its value for early diagnosis of schistosorniasis. Results At the 17th, 21st and 24th day after infection, the positive rates of antibody IgG of rabbits sera against GST-HD were 42.85%, 92.80% and 100.00% respectively, but the positive rates of antibody IgG against SEA were 14. 28% , 50. 00% and 84.60% respectively. The sensitivity of GST-HD for detecting early schistosome infection was higher than that of SEA significantly. The predictive values of positive and negative of GST-HD for detecting acute schistosorniasis was 98. 89% and 96.67%,respectively, and the diagnostic efficacy was 98. 33%. Conclusion The recombinant GST-HD fusion protein has high early diagnostic value for schistosorniasis.
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Objective To observe the molluscicidal effect of 25% suspension concentrate of niclosamide (SCN) against snail eggs and young snails. Methods The experiments with SCN and 50% wettable powder of niclosamide ethanolamide salt (WPN) against the snail eggs and young snails were carried out by the immersion method in laboratory. Results The death rates of snail eggs were both 100% in 0.25 mg/L active content of SCN and in 0.50 mg/L of WPN for 24 hours. The LC_~50(s) of SCN against Oncomelania snail eggs were 0.0506, 0.0496 mg/L and 0.0473 mg/L by immersion for 24, 48 hours and 72 hours respectively, while the LC_~50(s) of WPN were 0.1030, ~0.0962 mg/L and 0.0869 mg/L. The death rates of young snails were both 100% in 0.25 mg/L active content of SCN and WPN for 24 hours. The LC_~50(s) of SCN were 0.0625, 0.0474 mg/L and ~0.0442 mg/L for 24, 48 hours and 72 hours respectively, while the LC_~50(s) of WPN were 0.1088, 0.0825 ~mg/L and 0.0825 mg/L. Conclusion The SCN has high molluscicidal effect against Oncomelania snail in its different developmental stages: egg and young snail.[
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Objective To observe the impact of water temperature on shedding and infectivity of the cercariea of Schistosoma japonicum under laboratory condition. Methods Infected snails were exposed to 1, 5, 10, 15, 20, 25, 30, 35℃ and 40℃ water for 4 hours for shedding of cercariae respectively, the shedding rates and total numbers of cercariae shed were investigated. The mice were infected with cercariae in 1, 10, 20, 30℃ and 40℃ water for 30 minutes respectively, the infection rates and mean worm burden of mice were investigated. Results The cercariae were shed from infected snails with Jiangsu or Yunnan isolate of [WT5”BX]S. japonicum in water ranged from 1℃ to 40℃, but the fittest water temperatures range for shedding of cercariae of Yunnan isolate of S.japonicum[WT5”BZ] is from 20℃ to 35℃, while fittest range for Jiangsu isolate is from 15℃ to 35℃. All of the infection rates of mice infected with cercariae of Jiangsu isolate of S.japonicum in the water ranged from 1℃ to 40℃ were 100.0%. Infection rates of mice infected with cercariae of Yunnan isolate of S. japonicum in the water ranged from 1℃ to 40℃ were more than 83.3%. Conclusions When infected snails are exposed to the water with temperature ranged from 1℃ to 40℃ it is possible for cercariae to shed and infect the final hosts . It is suggested that there is a possibility of infection with S.japonicum when contacting the water near marshlands with infected snails in winter.
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Objective To develop a fast, simple immunodiagnosis assay for early diagnosis of schistosomiasis. Methods The soluble cercariae antigen(SCA) labeled colloidal dye was used as the detecting antigen for schistosomiasis. A dipstick dye immunoassay(SCA-DDIA) for early diagnosis of schistosomiasis was established. The antibodies in sera of infected rabbits in early stage of infection by SCA-DDIA were detected and compared with SEA-DDIA. The sera from people with acute and chronic schistosomiasis and other parasitic diseases and from healthy people were tested by SCA-DDIA and SEA-DDIA. Results In infected rabbits during early stage of infection, the average time of antibody detected by SCA-DDIA was 22 d , at day 30 post-infection all experimental rabbits were positive with SCA-DDIA, the detected time was earlier than that with SEA-DDIA. The sensitivity of SCA-DDIA for acute, chronic schistosomiasis japonica were 100.0% and 93.3% respectively. The specificity for healthy persons was 99.0%. The cross reaction rates with paragonimiasis westermani, clonorchiasis sinensis and fasciolopsiasis buski were 26.3%, 0 and 0 respectively. The results were similar to that by SEA-DDIA. Conclusion The SCA-DDIA is more useful for early diagnosis of schistosomiasis.
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Objective To understand the potential impact of south-north water transfer project on transmission and distribution of Schistosomiasis japonica, and to put forward the countermea-sures of prevention of the disease transferring into other places. Methods The information on the progress of south-north water transfer project and factors related to the distribution of Schistosoma juponicum were collected, and the suggestions on improving the countermeasures were obtained through the group discussions and field visits. Results The potential impact of the project on the disease transferring is existed, mainly the disease transferring will be through the Lixia River basin in Jiangsu Province, and Chaohu areas of Anhui Province in the east route, and Sihu areas of Hubei Province in the middle route. The snail transferring northward will be affected both by the project and global warming, as a result, the endemic areas of Schistosomiasis will probably transfer into the Hongzehu and Chaohu areas in the future. Conclusion In the east route of the project, if the project is not combined with Schistosomiasis control, the endemic areas of Schistosomiasis will extend into other regions, the loss in the society and economy will be very large.
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Objective To evaluate the effectiveness of the current measure of Oncomelania hupensis control in Jiangsu Province. Methods The snail control was carried out with molluscicides in the high transmission areas every year. Some snail habitat areas were modified. The snail areas within three years were re-treated with molluscicides in the maintenance phase. The snail survey was carried out every spring, and the data were analysed with SAS software. Results From 1995 to 2001, 14519.17 hm 2 of snail habitats were molluscicided, 2768.57 hm 2 were modified, and 8803.64 hm 2 were re-treated with molluscicides in the maintenance phase. The coverage rates of snail control areas dropped by 19.14% every year. The snail areas increased by 6.25% every year from 1995 to 2002. In which, the areas of infected snails increased by 18.52% every year. The correlation analysis showed that the areas of infected snails increased with the increasing of the total snail areas. At the same time, the areas of infected snails increased with the fall of the coverage rates of snail control areas. The analysis of the snail distribution showed that the main problem was poor snail control. Conclusion In recent years, the rise of area of snail habitats is serious in Jiangsu Province. The present measures of the snail control have not effectively stopped the spreading and increasing of snails. The research on the new molluscicides, the new methods of snail control and the better policies are very important.